Structural Insights into Retinal Guanylate Cyclase Activator Proteins (GCAPs) (original) (raw)
Related papers
2021
Neuronal calcium sensors play a crucial role in different pathways of Ca2+-mediated neurotransmission. Among them guanylate cyclase-activating protein 1 (GCAP1) is expressed only in photoreceptors and activates or inhibits retinal guanylate cyclase 1 (retGC1) depending on cellular Ca2+ concentrations during phototransduction. To date, 22 pathogenic mutations responsible for retinal dystrophy have been associated to GCAP1, but a complete picture of the molecular determinants of the disease is still missing. The only crystal structure available so far is the wt Ca2+-bound monomeric homologue from chicken and no cure exists for retinal dystrophy. In this work I report for the first time that the recombinant human GCAP1 is characterized by a highly dynamic monomer-dimer equilibrium, whose dissociation constant is influenced by salt concentration and by the nature of the divalent ion bound. Surprisingly, I discovered that also the chicken protein shows a similar mechanism, suggesting tha...
Human Molecular Genetics, 2015
Two recently identified missense mutations (p. L84F and p. I107T) in GUCA1A, the gene coding for guanylate cyclase (GC)activating protein 1 (GCAP1), lead to a phenotype ascribable to cone, cone-rod and macular dystrophies. Here, we present a thorough biochemical and biophysical characterization of the mutant proteins and their distinct molecular features. I107T-GCAP1 has nearly wild-type-like protein secondary and tertiary structures, and binds Ca 2+ with a >10-fold lower affinity than the wild-type. On the contrary, L84F-GCAP1 displays altered tertiary structure in both GC-activating and inhibiting states, and a wild type-like apparent affinity for Ca 2+. The latter mutant also shows a significantly high affinity for Mg 2+ , which might be important for stabilizing the GC-activating state and inducing a cooperative mechanism for the binding of Ca 2+ , so far not been observed in other GCAP1 variants. Moreover, the thermal stability of L84F-GCAP1 is particularly high in the Ca 2+-bound, GC-inhibiting state. Molecular dynamics simulations suggest that such enhanced stability arises from a deeper burial of the myristoyl moiety within the EF1-EF2 domain. The simulations also support an allosteric mechanism connecting the myristoyl moiety to the highestaffinity Ca 2+ binding site EF3. In spite of their remarkably distinct molecular features, both mutants cause constitutive activation of the target GC at physiological Ca 2+. We conclude that the similar aberrant regulation of the target enzyme results from a similar perturbation of the GCAP1-GC interaction, which may eventually cause dysregulation of both Ca 2+ and cyclic GMP homeostasis and result in retinal degeneration.
Human Molecular Genetics, 2015
Two recently identified missense mutations (p. L84F and p. I107T) in GUCA1A, the gene coding for guanylate cyclase (GC)activating protein 1 (GCAP1), lead to a phenotype ascribable to cone, cone-rod and macular dystrophies. Here, we present a thorough biochemical and biophysical characterization of the mutant proteins and their distinct molecular features. I107T-GCAP1 has nearly wild-type-like protein secondary and tertiary structures, and binds Ca 2+ with a >10-fold lower affinity than the wild-type. On the contrary, L84F-GCAP1 displays altered tertiary structure in both GC-activating and inhibiting states, and a wild type-like apparent affinity for Ca 2+. The latter mutant also shows a significantly high affinity for Mg 2+ , which might be important for stabilizing the GC-activating state and inducing a cooperative mechanism for the binding of Ca 2+ , so far not been observed in other GCAP1 variants. Moreover, the thermal stability of L84F-GCAP1 is particularly high in the Ca 2+-bound, GC-inhibiting state. Molecular dynamics simulations suggest that such enhanced stability arises from a deeper burial of the myristoyl moiety within the EF1-EF2 domain. The simulations also support an allosteric mechanism connecting the myristoyl moiety to the highestaffinity Ca 2+ binding site EF3. In spite of their remarkably distinct molecular features, both mutants cause constitutive activation of the target GC at physiological Ca 2+. We conclude that the similar aberrant regulation of the target enzyme results from a similar perturbation of the GCAP1-GC interaction, which may eventually cause dysregulation of both Ca 2+ and cyclic GMP homeostasis and result in retinal degeneration.
Journal of Biological Chemistry, 2019
The guanylyl cyclase-activating protein, GCAP1, activates photoreceptor membrane guanylyl cyclase (RetGC) in the light, when free Ca 2؉ concentrations decline, and decelerates the cyclase in the dark, when Ca 2؉ concentrations rise. Here, we report a novel mutation, G86R, in the GCAP1 (GUCA1A) gene in a family with a dominant retinopathy. The G86R substitution in a "hinge" region connecting EF-hand domains 2 and 3 in GCAP1 strongly interfered with its Ca 2؉-dependent activatorto-inhibitor conformational transition. The G86R-GCAP1 variant activated RetGC at low Ca 2؉ concentrations with higher affinity than did the WT GCAP1, but failed to decelerate the cyclase at the Ca 2؉ concentrations characteristic of darkadapted photoreceptors. Ca 2؉-dependent increase in Trp 94 fluorescence, indicative of the GCAP1 transition to its RetGC inhibiting state, was suppressed and shifted to a higher Ca 2؉ range. Conformational changes in G86R GCAP1 detectable by isothermal titration calorimetry (ITC) also became less sensitive to Ca 2؉ , and the dose dependence of the G86R GCAP1-RetGC1 complex inhibition by retinal degeneration 3 (RD3) protein was shifted toward higher than normal concentrations. Our results indicate that the flexibility of the hinge region between EF-hands 2 and 3 is required for placing GCAP1-regulated Ca 2؉ sensitivity of the cyclase within the physiological range of intracellular Ca 2؉ at the expense of reducing GCAP1 affinity for the target enzyme. The disease-linked mutation of the hinge Gly 86 , leading to abnormally high affinity for the target enzyme and reduced Ca 2؉ sensitivity of GCAP1, is predicted to abnormally elevate cGMP production and Ca 2؉ influx in photoreceptors in the dark. Guanylyl cyclase-activating proteins (GCAPs), 4 N-myristoylated calcium/magnesium-binding proteins of the EF-hand superfamily, are comprised of two pairs of EF-hand domains connected via a "hinge" region (reviewed in Refs. 1 and 2). Among several isoforms of GCAPs expressed in the vertebrate retinas (3-6) two, GCAP1 and GCAP2, regulate visual signaling in all species by properly shaping the sensitivity and kinetics of rod and cone responses (7-10). Vertebrate rods and cones respond to light stimuli by closing cGMP-gated channels in their outer segments via phototransduction cascade-mediated hydrolysis of cGMP (reviewed in Refs. 11 and 12). Following the excitation, cGMP production by retinal membrane guanylyl cyclase (RetGC) (13-15) first becomes accelerated, to speed up the recovery and light adaptation of photoreceptors, and then decelerated again as photoreceptors recover from the excitation back to their dark-adapted state (7, 16). Negative Ca 2ϩ feedback regulates the activity of RetGC via its Ca 2ϩ sensor proteins, GCAPs, such that in the light, when cGMP channels are closed and the influx of Ca 2ϩ through the channels stops, GCAPs release Ca 2ϩ and convert into a Mg 2ϩ-liganded state that stimulates RetGC. Once the photoreceptors return to their dark-adapted state, when cGMP channels reopen and the influx of Ca 2ϩ resumes, GCAPs undergo the reverse, activatorto-inhibitor, transition, by replacing Mg 2ϩ in their EF-hands with Ca 2ϩ , and decelerate RetGC (reviewed in Refs. 2 and 12). Failure of RetGC to accelerate or decelerate cGMP production within the normal range of the intracellular free Ca 2ϩ alters light sensitivity and kinetics of rod and cone response to light (7-9, 16-18) and has been linked to various forms of retinal blindness in humans, such as Leber congenital amaurosis, dominant cone or cone-rod degenerations (reviewed in Ref. 19-22), and a recessive night blindness (23). Multiple mutations linked to these blinding disorders have been found in the genes coding for RetGC1 isozyme (GUCY2D) (19-27) and GCAP1 (GUCA1A) (28-40). GUCA1A mutations linked to the domi
Retinal Guanylyl Cyclase-Activating Protein 1 and 2
Encyclopedia of Signaling Molecules, 2016
Historical Background Retinal guanylyl cyclases (RetGCs) in retinal rod and cone photoreceptors are regulated by a family of EF-hand Ca 2+ sensor proteins called guanylyl cyclase-activating proteins (GCAP1-8) that belong to the neuronal calcium sensor (NCS) family. Mammalian GCAPs (GCAP1 and GCAP2) activate RetGCs at low Ca 2+ levels in light-activated photoreceptor cells and inhibit RetGC activity at higher Ca 2+ levels in darkadapted photoreceptors. The Ca 2+-sensitive RetGC activity controlled by GCAPs is an important mechanism of visual recovery and light adaptation of phototransduction. Mutations in either RetGCs or GCAPs that disable this Ca 2+-sensitive cyclase activity are genetically linked to retinal disease. Here I review atomic-level structures of GCAP1 in both Ca 2+-free/Mg 2+-bound (activator) and Ca 2+-saturated (inhibitory) states, as well as the structure of Ca 2+-saturated GCAP2. The structure of GCAP2 reveals an exposed N-terminus that may be important for Ca 2+-dependent membrane anchoring of the myristoyl group. By contrast, the structures of Ca 2+-free and Ca 2+-bound forms of GCAP1 each contain a covalently attached myristoyl group that is sequestered in a hydrophobic protein cavity formed by helices at both the N-and C-terminus. Hence, myristoylated GCAP1 is not targeted to bilayer membranes. The Ca 2+-free activator form of GCAP1 contains Mg 2+ bound at the second EF-hand (EF2) that is essential for activating RetGC. The Ca 2+ saturated form of GCAP1 contains Ca 2+ bound at EF2, EF3, and EF4. Ca 2+-dependent conformational changes are most apparent in EF2 and in the Ca 2+ switch helix (residues 169-174) and will be discussed in terms of a proposed mechanism for Ca 2+-dependent activation of retinal guanylyl cyclases.
Frontiers in molecular neuroscience, 2014
Neuronal calcium sensor (NCS) proteins, a sub-branch of the calmodulin superfamily, are expressed in the brain and retina where they transduce calcium signals and are genetically linked to degenerative diseases. The amino acid sequences of NCS proteins are highly conserved but their physiological functions are quite different. Retinal recoverin controls Ca(2) (+)-dependent inactivation of light-excited rhodopsin during phototransduction, guanylyl cyclase activating proteins 1 and 2 (GCAP1 and GCAP2) promote Ca(2) (+)-dependent activation of retinal guanylyl cyclases, and neuronal frequenin (NCS-1) modulates synaptic activity and neuronal secretion. Here we review the molecular structures of myristoylated forms of NCS-1, recoverin, and GCAP1 that all look very different, suggesting that the attached myristoyl group helps to refold these highly homologous proteins into different three-dimensional folds. Ca(2) (+)-binding to both recoverin and NCS-1 cause large protein conformational c...
Activation of Retinal Guanylyl Cyclase-1 by Ca2+-binding Proteins Involves Its Dimerization
Journal of Biological Chemistry, 1999
Retinal guanylyl cyclase-1 (retGC-1), a key enzyme in phototransduction, is activated by guanylyl cyclase-activating proteins (GCAPs) if [Ca 2؉ ] is less than 300 nM. The activation is believed to be essential for the recovery of photoreceptors to the dark state; however, the molecular mechanism of the activation is unknown. Here, we report that dimerization of retGC-1 is involved in its activation by GCAPs. The GC activity and the formation of a 210-kDa cross-linked product of retGC-1 were monitored in bovine rod outer segment homogenates, GCAPs-free bovine rod outer segment membranes and recombinant bovine retGC-1 expressed in COS-7 cells. In addition to recombinant bovine GCAPs, constitutively active mutants of GCAPs that activate retGC-1 in a [Ca 2؉ ]-independent manner and bovine brain S100b that activates retGC-1 in the presence of ϳ10 M [Ca 2؉ ] were used to investigate whether these activations take place through a similar mechanism, and whether [Ca 2؉ ] is directly involved in the dimerization. We found that a monomeric form of retGC-1 (ϳ110 kDa) was mainly observed whenever GC activity was at basal or low levels. However, the 210-kDa product was increased whenever the GC activity was stimulated by any Ca 2؉ -binding proteins used. We also found that [Ca 2؉ ] did not directly regulate the formation of the 210-kDa product. The 210-kDa product was detected in a purified GC preparation and did not contain GCAPs even when the formation of the 210-kDa product was stimulated by GCAPs. These data strongly suggest that the 210-kDa cross-linked product is a homodimer of retGC-1. We conclude that inactive retGC-1 is predominantly a monomeric form, and that dimerization of retGC-1 may be an essential step for its activation by active forms of GCAPs.
Frontiers in Molecular Neuroscience
Over 100 mutations in GUCY2D that encodes the photoreceptor guanylate cyclase GC-E are known to cause two major diseases: autosomal recessive Leber congenital amaurosis (arLCA) or autosomal dominant cone-rod dystrophy (adCRD) with a poorly understood mechanism at the molecular level in most cases. Only few mutations were further characterized for their enzymatic and molecular properties. GC-E activity is under control of neuronal Ca 2+-sensor proteins, which is often a possible route to dysfunction. We investigated five recently-identified GC-E mutants that have been reported in patients suffering from arLCA (one large family) and adCRD/maculopathy (four families). Microsatellite analysis revealed that one of the mutations, c.2538G > C (p.K846N), occurred de novo. To better understand the mechanism by which mutations that are located in different GC-E domains develop different phenotypes, we investigated the molecular consequences of these mutations by expressing wildtype and mutant GC-E variants in HEK293 cells. Analyzing their general enzymatic behavior, their regulation by Ca 2+ sensor proteins and retinal degeneration protein 3 (RD3) dimerization domain mutants (p.E841K and p.K846N) showed a shift in Ca 2+-sensitive regulation by guanylate cyclase-activating proteins (GCAPs). Mutations in the cyclase catalytic domain led to a loss of enzyme function in the mutant p.P873R, but not in p.V902L. Instead, the p.V902L mutation increased the guanylate cyclase activity more than 20fold showing a high GCAP independent activity and leading to a constitutively active mutant. This is the first mutation to be described affecting the GC-E catalytic core in a complete opposite way.
Human Mutation, 2009
The GUCA1A gene encodes the guanylate cyclase activating protein 1 (GCAP1) of mammalian rod and cone photoreceptor cells, which is involved in the Ca 2+ -dependent negative feedback regulation of membrane bound guanylate cyclases in the retina. Mutations in the GUCA1A gene have been associated with different forms of cone dystrophies leading to impaired cone vision and retinal degeneration. Here we report the identification of three novel and one previously detected GUCA1A mutations: c.265G>A (p.Glu89Lys), c.300T>A (p.Asp100Glu), c.476G>T (p.Gly159Val) and c.451C>T (p.Leu151Phe). The clinical data of the patients carrying these mutations were compared with the functional consequences of the mutant GCAP1 forms. For this purpose we purified the heterologously expressed GCAP1 forms and investigated whether the mutations affected the Ca 2+ -triggered conformational changes and the apparent interaction affinity with the membrane bound guanylate cyclase. Furthermore, we analyzed Ca 2+ -dependent regulatory modes of wildtype and mutant GCAP1 forms. Although all novel mutants were able to act as a Ca 2+ -sensor protein, they differed in their Ca 2+ -dependent activation profiles leading to a persistent stimulation of guanylate cyclase activities at physiological intracellular Ca 2+ concentration.
Guanylate cyclases and associated activator proteins in retinal disease
2010
Two isoforms of guanylate cyclase, GC1 and GC2 encoded by GUCY2D and GUCY2F, are responsible for the replenishment of cGMP in photoreceptors after exposure to light. Both are required for the normal kinetics of photoreceptor sensitivity and recovery, although disease mutations are restricted to GUCY2D. Recessive mutations in this gene cause the severe early-onset blinding disorder Leber congenital amaurosis whereas dominant mutations result in a later onset less severe cone-rod dystrophy. Cyclase activity is regulated by Ca 2? which binds to the GC-associated proteins, GCAP1 and GCAP2 encoded by GUCA1A and GUCA1B, respectively. No recessive mutations in either of these genes have been reported. Dominant missense mutations are largely confined to the Ca 2?binding EF hands of the proteins. In a similar fashion to the disease mechanism for the dominant GUCY2D mutations, these mutations generally alter the sensitivity of the cyclase to inhibition as Ca 2? levels rise following a light flash.